Abstract 2598: Fatty origins of a bone tumor? The curious case of Ewing Sarcoma

Abstract Ewing sarcoma (EwS) is an aggressive pediatric bone tumor with an enigmatic cell-of-origin. The fusion oncogene EWS::FLI1 is pathognomonic for EwS and the primary driver in 85% of EwS patients. Human mesenchymal stem cells (hMSCs) are considered to be the likely cell of origin of EwS for two reasons: their ability to tolerate ectopic EWS::FLI1 expression and the resemblance in the transcriptomes of EWS::FLI1 expressing hMSCs to primary Ewing tumors. However, to date a cellular differentiation-based model of EwS does not exist. To obtain insights into EwS etiology and histogenesis, this study has established the first comprehensive single-cell differentiation atlas of juvenile bone marrow-derived hMSCs with and without EWS::FLI1 expression. A pool of bone-marrow derived hMSCs from nineteen healthy juvenile donors was engineered to conditionally express EWS::FLI1. We used physiology mimicking conditions (hypoxia, spheroid cultures, serum-free growth and differentiation media) and induced EWS::FLI1 oncogene expression for 24h or 72h at daily time points of hMSC differentiation into adipo-, chondro-, osteo- and neuro-genic lineages. This highly resolved atlas of 252 samples representing normal and perturbed differentiation allowed us to map single cell tumor data from EwS patients to multiple lineages and time-points across stages of lineage commitment. Single-cell RNA-seq analysis of ∼488, 000 transcriptomes revealed loss of lineage identity in the presence of EWS::FLI1 across all four lineages. However, the analysis of genes detected in the EWS::FLI1 high cluster uncovered a specific time and lineage preference towards treated adipogenic cells that shared the transcriptomic signature with tumor cells, indicating the potential cell/stage of tumor-origin. Longer EWS::FLI1 induction led to enhanced loss of lineage identity and higher mapping with patient data. Soft agar colony formation assay confirmed stronger transformation potential of EWS::FLI1 expressing adipogenic cells. Our results offer novel insights into the elusive cellular origins of Ewing sarcoma, thereby establishing a foundation for the development of indispensable preclinical models to inform tailored therapeutic approaches. This study is supported by a generous grant (#20-17258) of the Alex’s Lemonade Stand Foundation (ALSF) Crazy8 Program. Citation Format: Utkarsh Kapoor, Christoph Hafemeister, Lisa Shaw, Karin Muelbacher, Julia Moldaschl, Caterina Sturtzel, Dominik Egger, Stefan Toegel, Cornelia Kasper, Martin Distel, Kelly M. Bailey, Marc van de Wetering, Masanori Hayashi, Matthias Farlik, Florian Halbritter, Heinrich Kovar. Fatty origins of a bone tumor? The curious case of Ewing Sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2598.